The long term goal of this research is to identify and characterize intracellular sites of volatile anesthetic binding, and subsequently determine the relevance to anesthesia. The hypothesis this proposal addresses is that halothane binds specifically to intracellular sites in the nematode, C. elegans, and that this binding is altered in mutant nematodes with increased halothane sensitivity (unc-79 & unc-80). Two techniques will be used to address this hypothesis. First, the subcellular distribution of halothane will be determined in C. elegans with a radiolysis/electron probe microanalysis (EPMA) method developed in our laboratory. Correlations between sensitivity and distribution will be examined in 4 different phenotypes of C. elegans. Preliminary results demonstrate that subcellular halothane distribution in the nematode is similar to that of rat (previous study), but that significant differences in distribution do exist between the phenotypes. Alterations in halothane distribution with the introduction of other volatile agents (competition) will be examined. the binding/distribution of halothane will be examined at higher resolution with a photochemical technique. Radiolabeled halothane will be incubated with various subfractions of nematodes, and fixed with UV light. Preliminary results show that 14C- halothane binds specifically to a crude membrane preparations, but that this binding is similar between the three of the nematode phenotypes examined. Specificity of binding appears to follow sensitivity of the intact animal to several different anesthetics. the specificity of binding will be determined with displacement and saturation experiments, and the molecular location of label can be determined with conventional biochemical separative methods. The relevance to anesthesia will be examined by a comparison of location and binding characteristics between the 4 phenotypes. These studies represent a novel approach to the site of anesthetic action, heretofore impossible because of the high volatility of the agents, rapid kinetics, absence of antagonists and apparent low affinity.